PROJECT SUMMARY The overall goal of this proposal is to determine the genetic variants that alter complex three-dimensional morphologies and to determine how these genes exert their influence on cell number, shape and behavior during development. Developmental genes that are important in morphogenesis are linked in complex hierarchies, yet we do not currently know how to prioritize variants within the context of a gene regulatory network. I will therefore undertake an evolutionary case study at the level of a gene regulatory network to determine the types of genes that contribute to morphological diversification between species and characterize their effects on individual cells. To accomplish this goal, I require a system where the gene regulatory network underlying a quantitative trait is well-understood, and where critical variants can be mapped at the resolution of individual genes. The posterior lobe in the Drosophila melanogaster clade is particularly well-suited to connecting genetic variation to morphogenetic outcomes. Recent work has identified many components of the gene regulatory network controlling posterior lobe development, including patterning molecules (transcription factors and signaling molecules) and cellular effectors (cytoskeletal regulators). Furthermore, hybrids between all three species are viable and exhibit intermediate lobe morphologies, which allows for rigorous mapping of genomic regions contributing to their drastically different phenotypes. In Aim 1, I will map sequences responsible for quantitative variation in posterior lobe morphology. I will use RNA-seq in species and hybrids to identify genes containing expression variation, with the expectation that some of these genes contribute to morphological diversity in the posterior lobe. I will then validate strong candidates using a CRISPR-based complementation test. In Aim 2, I will determine how patterning molecules and cellular effectors influence the number, shape and behavior of individual cells. I will use cell labeling and imaging to measure cell number, size and behavior in posterior lobes derived from mutants, different species and species hybrids. By completing this proposal, I will connect variation in the gene regulatory network to molecular mechanisms that dictate cell size and shape, which will develop the posterior lobe as a premier system for systems-level studies of morphological development and evolution.